The present invention generally relates to a method of regulating transforming growth factor TGF-xcex2 (TGF-xcex2) activity. More particularly, the present invention relates to a method of interfering with the activation of TGF-xcex2 by thrombospondin through the administration of peptides in order to treat kidney diseases and/or conditions.
Extracellular matrix accumulation is one of the hallmarks of inflammatory glomerular disease as a major cause of end-stage renal disease in man. Mesangial proliferative glomerulonephritis, the most common type of glomerulonephritis in the Western World (D""Amico (1987) QJM 245, 709-727), is characterized by mesangial cell (MC) proliferation, activation, and extracellular matrix expansion (Johnson (1994) Kidney Int. 45 (6), 1769-82). In up to 50% of the patients with mesangial proliferative glomerulonephritis, the disease process eventually progresses to end-stage renal disease, since specific treatment is still lacking (Galla (1995) Kidney Int. 47, 377-387). Typical features of human mesangial proliferative glomerulonephritis are mimicked by an experimental model in the rat, induced by an antibody against the Thy1-antigen on MC ((Johnson (1994) Kidney Int. 45 (6), 1769-82). In this model, a single injection of anti-thymocyte antibody results in an acute, complement-dependent MC injury (days zero to two) with proteinuria, followed by a FGF-2- and PDGF-dependent MC proliferative response that is accompanied by a TGF-xcex2-dependent overproduction of extracellular matrix proteins (days three to ten) (Johnson (1994) Kidney Int. 45 (6), 1769-82).
The role of TGF-xcex2 as a major profibrotic cytokine in the anti-Thy1 model has been well established (Border et al. (1994) N. Engl. J. Med. 331, 1286-1292). It has been demonstrated that TGF-xcex21 mRNA and protein are increased in the anti-Thy1 model (Okuda et al. (1990) J. Clin. Invest. 86, 453-462) and that blocking TGF-xcex21 by injections with a polyclonal anti-TGF-xcex21 antibody markedly reduced extracellular matrix accumulation (Border et al. (1990) Nature 346, 371-374). Injections with the proteoglycan decorin, a TGF-xcex21, -2, and -3 binding protein, also suppressed TGF-xcex2-dependent alterations such as extracellular matrix accumulation in the anti-Thy1 model (Border et al. (1992) Nature 360, 361-364). The results of these studies by Border and colleagues were confirmed by studies using gene transfer techniques in the anti-Thy1 model. Transfer of antisense oligonucleotides against the TGF-xcex21 mRNA into the rat kidney suppressed upregulation of glomerular TGF-xcex21 mRNA and protein as well as extracellular matrix accumulation (Akaki et al. (1996) Kidney Int. 50, 148-155). Transfer of decorin cDNA into rat skeletal muscle increased the amount of decorin in skeletal muscle and in the kidney, and again ameliorated glomerular disease by decreasing matrix formation (Isaka et al. (1996) Nat. Med. 2, 418-423). In contrast, mice transgenic for an active form of TGF-xcex21 exhibit elevated plasma levels of TGF-xcex21 and develop progressive renal disease characterized by MC matrix accumulation, interstitial fibrosis, and proteinuria (Kopp et al. (1996) Lab. Invest. 74, 991-1003). Transfer of the TGF-xcex21 gene into glomeruli of normal rats caused an increase in glomerular TGF-xcex21 protein that was linked to extracellular matrix formation (Isaka et al (1993) J. Clin. Invest. 92, 2597-2601). The potential importance of TGF-xcex2 in mediating fibrosis also in human kidney disease has been supported by the widespread link of TGF-xcex2 upregulation and extracellular matrix excess in many different types of human kidney disease (Border et al. (1994) N. Engl. J. Med. 331, 1286-1292).
While these studies suggest great benefit from suppression of TGF-xcex2 function in fibrotic kidney disease, it has to be considered that TGF-xcex2 is a multifunctional cytokine that exhibits other essential functions in mammals. Mice lacking either the TGF-xcex21, or -2, or -3 gene do not survive beyond a few weeks after birth (Shull et al. (1992) Nature 359, 693-699; Sanford et al. (1997) Development 124, 2659-2670; Kaartinen et al. (1995) Nature Genet. 11, 415-421). TGF-xcex21 null mice die a few weeks after birth from a severe generalized inflammatory response demonstrating that complete suppression of TGF-xcex2 function must not be a therapeutic goal in treating inflammatory kidney disease (Shull et al. (1992) Nature 359, 693-699). Therefore, accurate regulation of TGF-xcex2 function seems to be critical for the health of mammals and any anti-TGF-xcex2 therapeutic approach should try to target the local overproduction (-function) of TGF-xcex2 as specifically as possible.
One possibility to approach this goal could be by controlling (interfering with) the activation process of locally produced TGF-xcex2. TGF-xcex2 is secreted by most cell types as a latent, inactive procytokine-complex that consists of the mature, active TGF-xcex2 protein, which is noncovalently bound to a dimer of its N-terminal propeptide, the so-called latency-associated protein (LAP), and variably to a latent TGF-xcex2 binding protein (LTBP) (Harpel et al. (1992) Prog. Growth Factor Res. 4, 321-335). The mature TGF-xcex2 protein has to be extracellularly released or unmasked from this procytokine-complex to be able to interact with its receptors. While various players/mechanisms such as pH changes, gamma irradiation, reactive oxygen species, plasmin, calpain, cathepsin, or thrombospondin 1 (TSP1) have been identified to activate TGF-xcex2 under in vitro conditions, it is still unknown how TGF-xcex2 is activated in an inflammatory process in vivo (Harpel et al. (1992) Prog. Growth Factor Res. 4, 321-335).
Recent data have suggested the homotrimeric extracellular matrix protein TSP1 as an activator of TGF-xcex21 in vitro in different cell systems including MC (Schultz-Cherry et al. (1993) J. Cell Biol. 122, 923-932; Tada et al. (1998) Nephron 79, 38-43; Schultz-Cherry et al. (1994) J. Biol. Chem. 269, 26775-82) as well as in cell free systems. It has been demonstrated that TSP1 forms a trimolecular complex with the TGF-xcex2 procytokine-complex by interacting with the mature TGF-xcex2 protein as well as the LAP (Ribeiro et al. (1999) J. Biol. Chem. 274, 13586-13593). Hereby, the hexapeptide (AA or GG) WSHW (SEQ ID NO:22 or SEQ ID NO:3, respectively) from the type I repeats of the TSP1 molecule is required for TSP1-binding to the mature TGF-xcex2 protein facilitating interaction of the KRFK-amino acid sequence (SEQ. ID NO:5) of the TSP1 molecule with the N-terminal LSKL-sequence (SEQ ID NO:21) of the LAP (Schultz-Cherry et al. (1995) J. Biol. Chem. 270, 7304-7310; Ribeiro et al. (1999) J. Biol. Chem. 274, 13586-13593). This complex interaction is thought to induce a conformational change probably within the LAP that allows the mature TGF-xcex2 protein to bind to its receptors. It has been shown that both the hexapeptide AAWSHW (SEQ. ID NO:22) and the LSKL (SEQ. ID NO:21) peptides are able to block activation of TGF-xcex2 by TSP1. In addition, comparing TSP1 null mice with TGF-xcex21 null mice, Crawford et al. identified TSP1 as a major activator of TGF-xcex21 in vivo during mouse post-natal development (Crawford et al. (1998) Cell 93, 1159-1170). Organ pathology of TGF-xcex21 null pups and TSP1 null pups were strikingly similar and could be induced in wild type pups by intraperitoneal (i.p.) treatment with the LSKL-peptide that specifically blocks activation of TGF-xcex21 by TSP1. Loss of TSP1 expression in TSP1 null mice spontaneously produced inflammatory lung disease (Lawler et al. (1998) J. Clin. Invest. 101, 982-992) and histological changes in TSP1 null mice reverted toward wild type by treatment with the TGF-xcex2 activating peptide KRFK (SEQ ID NO:5).
Interestingly, TSP1 expression in vitro is regulated by various cytokines such as PDGF, FGF-2, or TGFxcex2, and is frequently expressed de novo at sites of inflammation and wound heating (Bornstein (1995) J. Cell Biol. 130, 503-506). The involvement of TSP1 in the anti-Thy1 model has been demonstrated (Hugo et al. (1995) Kidney Int. 48, 1846-1856). In the anti-Thy1 model of mesangial proliferative glomerulonephritis, a marked transient de novo expression of TSP1 by MC (peak on day five) was regulated by FGF-2 and PDGF (Hugo et al. (1995) Kidney Int. 48, 1846-1856) and coincided with the upregulation of TGF-xcex21 (Okuda et al. (1990) J. Clin. Invest. 86, 453-462).
Therefore, it is hypothesized that TSP1 is a major endogenous activator of TGF-xcex2 in inflammatory kidney disease and it was investigated whether systemic treatment with either one of two different peptides that block activation of TGF-xcex21 by TSP1 is able to suppress activation and function of TGF-xcex2 in experimental mesangial proliferative nephritis in the rat.
Accordingly, it would be advantageous and desirable to have a method for treating inflammatory kidney diseases, including mesangial proliferative glomerulonephritis, by treating a subject in need of such treatment with a peptide capable of blocking the activation of TGF-xcex21.
The present invention provides a method of treating kidney or renal diseases/conditions in a subject by administering to the subject a pharmaceutically effective amount of a purified peptide including the LAP domain peptide, the TSP-1 type 1 domain peptide, or fragments thereof to interfere with the activation process of TGF-xcex2 to reduce and/or prevent renal damage.
The present invention further provides a method of improving renal function in a subject having impaired renal function by administering to the subject a pharmaceutically effective amount of a purified peptide including the LAP domain peptide, the TSP-1 type 1 domain peptide, or fragments thereof